Compositions and methods for cancer immunotherapy

ABSTRACT

The present invention provides a combination therapy which relies on a small molecule immune stimulator—cyclic-di-nucleotide (CDN)—that activates DCs via a recently discovered cytoplasmic receptor known as STING (Stimulator of Interferon Genes) formulated with allogeneic human tumor cell lines engineered to secrete high amounts of GM-CSF. This combination therapy can provide an ideal synergy of multiple tumor associated antigens, DC recruitment and proliferation, coupled with a potent DC activation stimulus.

The present application claims priority to U.S. Provisional PatentApplication 61/657,574 filed Jun. 8, 2012, which is hereby incorporatedin its entirety, including all tables, figures, and claims

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

The human immune system may generally be divided into two arms, referredto as “innate immunity” and “adaptive immunity.” The innate arm of theimmune system is predominantly responsible for an initial inflammatoryresponse via a number of soluble factors, including the complementsystem and the chemokine/cytokine system; and a number of specializedcell types including mast cells, macrophages, dendritic cells (DCs), andnatural killer cells. In contrast, the adaptive immune arm involves adelayed and a longer lasting antibody response together with CD8+ andCD4+ T cell responses that play a critical role in immunological memoryagainst an antigen. A third arm of the immune system may be identifiedas involving γδ T cells and T cells with limited T cell receptorrepertoires such as NKT cells and MAIT cells.

For an effective immune response to an antigen, antigen presenting cells(APCs) must process and display the antigen in a proper MHC context to aT cell, which then will result in either T cell stimulation of cytotoxicand helper T cells. Following antigen presentation successfulinteraction of co-stimulatory molecules on both APCs and T cells mustoccur or activation will be aborted. GM-CSF and IL-12 serve as effectivepro-inflammatory molecules in many tumor models. For example, GM-CSFinduces myeloid precursor cells to proliferate and differentiate intodendritic cells (DCs) although additional signals are necessary toactivate their maturation to effective antigen-presenting cellsnecessary for activation of T cells. Barriers to effective immunetherapies include tolerance to the targeted antigen that can limitinduction of cytotoxic CD8 T cells of appropriate magnitude andfunction, poor trafficking of the generated T cells to sites ofmalignant cells, and poor persistence of the induced T cell response.

DCs that phagocytose tumor-cell debris process the material for majorhistocompatibility complex (MHC) presentation, upregulate expression ofcostimulatory molecules, and migrate to regional lymph nodes tostimulate tumor-specific lymphocytes. This pathway results in theproliferation and activation of CD4+ and CD8+ T cells that react totumor-associated antigens. Indeed, such cells can be detected frequentlyin the blood, lymphoid tissues, and malignant lesions of patients.

New insights into the mechanisms underlying immune-evasion, togetherwith combination treatment regimens that potentiate the potency oftherapeutic vaccination—either directly or indirectly—throughcombination with immune checkpoint inhibitors or other therapies, haveserved as a basis for the development of vaccines that induce effectiveantitumor immunity.

Tumor cells genetically modified to secrete GM-CSF have been used invarious strategies in an effort to generate an effective immune responseto tumors, however systemic cytokine administration has not induced adirect anti-cancer response in randomized controlled trials. IrradiatedGM-CSF-secreting tumor cells injected subcutaneously into patients havebeen shown to stimulate a local response comprising DCs, macrophages,and granulocytes. The accumulation of large numbers of APCs suggeststhat one function of GM-CSF in this model involved the augmentation oftumor antigen presentation. Moreover, tumor cell vaccines have shown tobe safe in patients. However, the clinical efficacy of this approach hasbeen yet to be proven.

In the context of infection, Toll-like receptor (“TLR”) agonists havebeen shown to render dendritic cell activation immunogenic, whereas lackof TLR signaling can lead to tolerance. The implication from thesestudies is that localized TLR stimulation might enhance antitumorresponse when given as part of a combinatorial vaccine. WO2011139769describes the formulation and use of a combined GM-CSF-secreting tumorcell (GVAX) vaccine, together with TLR4 stimulation, which reportedlyprovided anti-tumor efficacy in several murine models. Its efficacy inhumans remains, however, to be proven.

There remains a need for improved compositions and methods forimmunologic strategies to treating diseases such as cancer that can berefractory to traditional therapeutic approaches.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide combinationtherapies for the treatment of cancer.

In a first aspect, the present invention provides compositionscomprising:

one or more cyclic purine dinucleotides (“CDN”) which binds toSTimulator of INTerferon Gene (“STING”) and induces STING-dependent TBK1activation; and an inactivated tumor cell which expresses and secretesone or more cytokines which stimulate dendritic cell induction,recruitment and/or maturation.

As described hereinafter, a number of CDNs find use in the presentinvention. Preferred cyclic purine dinucleotides include, but are notlimited to, one or more of c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP,c-AMP-IMP, c-GMP-IMP, and analogs thereof. This list is not meant to belimiting.

Similarly, preferred costimulatory agent comprises one or more cytokineswhich stimulate dendritic cell induction, recruitment, and/or maturationinclude, but are not limited to, one or more of GM-CSF, CD40 ligand,IL-12, CCL3, CCL20, and CCL21. This list is not meant to be limiting.

The compositions of the present invention may be administered toindividuals in need thereof by a variety of parenteral and nonparenteralroutes in formulations containing pharmaceutically acceptable carriers,adjuvants and vehicles. Preferred routes are parenteral, and includebut, are not limited to, one or more of subcutaneous, intravenous,intramuscular, intraarterial, intradermal, intrathecal and epiduraladministrations. Particularly preferred is administration bysubcutaneous administration. Preferred pharmaceutical composition areformulated as aqueous or oil-in-water emulsions.

The compositions of the present invention may comprise, or beadministered together with, one or more additional pharmaceuticallyactive components such as adjuvants, lipids such as digitonin,liposomes, CTLA-4 and PD-1 pathway Antagonists, PD-1 pathway blockingagents, inactivated bacteria which induce innate immunity (e.g.,inactivated or attenuated Listeria monocytogenes), compositions whichmediate innate immune activation via Toll-like Receptors (TLRs),(NOD)-like receptors (NLRs), Retinoic acid inducible gene-based(RIG)-1-like receptors (RLRs), C-type lectin receptors (CLRs),pathogen-associated molecular patterns (“PAMPs”), chemotherapeuticagents, etc.

As described hereinafter, cyclic purine dinucleotides formulated withone or more lipids can exhibit improved properties, including improveddendritic cell activation activity. Thus, the present invention alsorelates to a composition comprising one or more CDNs and one or morelipids. In certain preferred embodiments, one or more CDNs areformulated with digitonin, a liposomal formulation, and/or anoil-in-water emulsion. While these formulations of the invention may beadministered without an inactivated tumor cell which expresses andsecretes one or more cytokines which stimulate dendritic cell induction,recruitment and/or maturation, in certain embodiments, the formulationof CDNs with one or more lipids are provided together with one or moresuch cell lines.

In related aspects, the present invention relates to methods forinducing an immune response to a cancer in an individual. These methodscomprise administering a composition according to the present inventionto an individual in need thereof, wherein the inactivated tumor cell ora mixture of different tumor cells are type-matched to the individual'scancer.

In certain embodiments, the inactivated tumor cells or a mixture ofdifferent tumor cells are allogeneic tumor cells or autologous tumorcells or a mixture of the two.

The methods of the present invention may be directed to patients beingtreated for colorectal cancer, an aero-digestive squamous cancer, a lungcancer, a brain cancer, a liver cancer, a stomach cancer, a sarcoma, aleukemia, a lymphoma, a multiple myeloma, an ovarian cancer, a uterinecancer, a breast cancer, a melanoma, a prostate cancer, a pancreaticcarcinoma, and a renal carcinoma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts cyclic purine dinucleotide (“CDN”)-mediated signaling. ACDN (e.g., c-di-AMP c-di-GMP, c-AMP-GMP), with the two purinenucleosides alternatively joined by a phosphate bridge with canonicalbis-(3′,5′) linkages, or non-canonical 2′,5′ and 3′,5′ linkages,represented by c[G(2′,5′)pA(3′,5′)p]. The canonical or non-canonicalCDNs induce production of both NF-kB dependent pro-inflammatorycytokines, and also IFN-β by binding to the cytosolic receptor STING(Stimulator of Interferon Genes), activating signaling through theTBK-1/IRF-3 pathway, resulting in both autocrine and paracrineactivation of DCs through binding to the IFN receptor and subsequentsignaling.

FIG. 2A depicts CDN-adjuvinated T-cell responsiveness to HIV Gag.

FIG. 2B depicts The primary and secondary OVA-specific CD4 and CD8 Tcell response in PBMC following immunization of mice with the vaccinecompositions shown in the figure.

FIG. 2C depicts immune responses induced by CDN-adjuvanted vaccinesusing OVA as a model antigen.

FIG. 3A depicts growth inhibition of B16 melanoma in response to aGVAX/CDN combination vaccine (referred to as “Stingvax”).

FIG. 3B depicts interferon-β induction in TRAMP-GM cells and bonemarrow-derived macrophages in response to a GVAX/CDN combinationvaccine.

FIG. 4 depicts concentration dependence of growth inhibition of B16melanoma in response to a GVAX/CDN combination vaccine.

FIG. 5A depicts CD8+ T-cell infiltration of untreated B16 melanomatumors.

FIG. 5B depicts CD8+ T-cell infiltration of B16 melanoma tumors in cellstreated with CDN.

FIG. 5C depicts CD8+ T-cell infiltration of B16 melanoma tumors in cellstreated with GVAX.

FIG. 5D depicts CD8+ T-cell infiltration of B16 melanoma tumors in cellstreated with CDN and GVAX.

FIG. 6 depicts induction of mature interferon γ-producing splenic DC(CD11c+ cells) in response to a GVAX/CDN combination vaccine.

FIG. 7 depicts the induction of human dendritic cells upon CDNtreatment, as assessed by expression of costimulatory molecules.

FIG. 8 depicts the expression of IFN-α in cultured human peripheralblood mononuclear cells isolated from 15 independent donors, followingstimulation with various activators of innate immunity, includingcyclic-di-GMP (CDG), Interferon Stimulating DNA (ISD), and polyinosine-cytosine (Poly I:C).

FIG. 9 depicts the synergistic mechanism of action of “STINGVAX.”

DETAILED DESCRIPTION OF THE INVENTION

The FDA approval of Provenge® (Dendreon Corporation) for the treatmentof castration-resistant metastatic prostate cancer (mCRPC) has validatedactive cancer immunotherapy as a therapeutic area and reinvigorated thefield. However, as an autologous dendritic cell (DC) based vaccine,Provenge® is impractical, complex and expensive, and provides only amodest—albeit significant—survival benefit. An improved cancer vaccineshould be not only at least as effective as Provenge®, but also morepractical, and preferably able to elicit objective responses. One stepin this direction is ProstVac VF, a recombinant pox virus-based vaccinethat encodes PSA and three co-stimulatory molecules (B7.1, ICAM-1, andLfa-3). ProstVac VF was shown to increase overall survival in arandomized Phase 2 study conducted among men with mCRPC—though survivalbenefit did not reach p<0.05 statistical significance—and is currentlybeing evaluated in a Phase 3 efficacy study. These vaccines use a singleantigen expressed by both normal prostate tissue and prostate cancer.Targeting multiple cancer antigens with therapeutic vaccinationstrategies is desirable to reduce the potential negative impact ofantigen-loss variants, patient-by-patient differences intumor-associated antigen expression profiles, or MHC haplotypedifferences, all issues with single TAA vaccination strategies.

The present invention relates to a novel and highly active combinationtherapy which relies on a small molecule immunestimulator—cyclic-di-nucleotide (CDN)—that activates DCs via a recentlydiscovered cytoplasmic receptor known as STING (Stimulator of InterferonGenes) formulated with irradiated allogeneic human tumor cell linesengineered to secrete high amounts of the DC growth factor, GM-CSF. Thiscombination therapy can provide an ideal synergy of multiple tumorassociated antigens, DC recruitment and proliferation (GM-CSF), coupledwith a potent DC activation stimulus (CDN).

The CDNs cyclic-di-AMP (produced by Listeria monocytogenes) and itsanalog cyclic-di-GMP (produced by Legionella pneumophila) are recognizedby the host cell as a PAMP (Pathogen Associated Molecular Pattern),which bind to the PRR (Pathogen Recognition Receptor) known as STING.STING is an adaptor protein in the cytoplasm of host mammalian cellswhich activates the TANK binding kinase (TBK1)-IRF3 signaling axis,resulting in the induction of IFN-β and other IRF-3 dependent geneproducts that strongly activate innate immunity. It is now recognizedthat STING is a component of the host cytosolic surveillance pathway,that senses infection with intracellular pathogens and in responseinduces the production of IFN-β, leading to the development of anadaptive protective pathogen-specific immune response consisting of bothantigen-specific CD4 and CD8 T cells as well as pathogen-specificantibodies.

Definitions

“Administration” as it is used herein with regard to a human, mammal,mammalian subject, animal, veterinary subject, placebo subject, researchsubject, experimental subject, cell, tissue, organ, or biological fluid,refers without limitation to contact of an exogenous ligand, reagent,placebo, small molecule, pharmaceutical agent, therapeutic agent,diagnostic agent, or composition to the subject, cell, tissue, organ, orbiological fluid, and the like. “Administration” can refer, e.g., totherapeutic, pharmacokinetic, diagnostic, research, placebo, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” also encompassesin vitro and ex vivo treatments, e.g., of a cell, by a reagent,diagnostic, binding composition, or by another cell. By “administeredtogether” it is not meant to be implied that two or more agents beadministered as a single composition. Although administration as asingle composition is contemplated by the present invention, such agentsmay be delivered to a single subject as separate administrations, whichmay be at the same or different time, and which may be by the same routeor different routes of administration.

By “purified” and “isolated” is meant that a specified species accountsfor at least 50%, more often accounts for at least 60%, typicallyaccounts for at least 70%, more typically accounts for at least 75%,most typically accounts for at least 80%, usually accounts for at least85%, more usually accounts for at least 90%, most usually accounts forat least 95%, and conventionally accounts for at least 98% by weight, orgreater, of the species present in a composition. The weights of water,buffers, salts, detergents, reductants, protease inhibitors, stabilizers(including an added protein such as albumin), and excipients aregenerally not used in the determination of purity.

“Specifically” or “selectively” binds, when referring to aligand/receptor, nucleic acid/complementary nucleic acid,antibody/antigen, or other binding pair (e.g., a cytokine to a cytokinereceptor) (each generally referred to herein as a “target biomolecule”or a “target”) indicates a binding reaction which is related to thepresence of the target in a heterogeneous population of proteins andother biologics. Specific binding can mean, e.g., that the bindingcompound, nucleic acid ligand, antibody, or binding composition derivedfrom the antigen-binding site of an antibody, of the contemplated methodbinds to its target with an affinity that is often at least 25% greater,more often at least 50% greater, most often at least 100% (2-fold)greater, normally at least ten times greater, more normally at least20-times greater, and most normally at least 100-times greater than theaffinity with a non-target molecule.

“Ligand” refers to a small molecule, nucleic acid, peptide, polypeptide,saccharide, polysaccharide, glycan, glycoprotein, glycolipid, orcombinations thereof that binds to a target biomolecule. While suchligands may be agonists or antagonists of a receptor, a ligand alsoencompasses a binding agent that is not an agonist or antagonist, andhas no agonist or antagonist properties. Specific binding of a ligandfor its cognate target is often expressed in terms of an “Affinity.” Inpreferred embodiments, the ligands of the present invention bind withaffinities of between about 10⁴ M⁻¹ and about 10⁸ M⁻¹. Affinity iscalculated as K_(d)=k_(off)/k_(on) (k_(off) is the dissociation rateconstant, K_(on) is the association rate constant and K_(d) is theequilibrium constant).

Affinity can be determined at equilibrium by measuring the fractionbound (r) of labeled ligand at various concentrations (c). The data aregraphed using the Scatchard equation: r/c=K(n−r): where r=moles of boundligand/mole of receptor at equilibrium; c=free ligand concentration atequilibrium; K=equilibrium association constant; and n=number of ligandbinding sites per receptor molecule. By graphical analysis, r/c isplotted on the Y-axis versus r on the X-axis, thus producing a Scatchardplot. Affinity measurement by Scatchard analysis is well known in theart. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelsonand Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988. In analternative, affinity can be measured by isothermal titrationcalorimetry (ITC). In a typical ITC experiment, a solution of ligand istitrated into a solution of its cognate target. The heat released upontheir interaction (ΔH) is monitored over time. As successive amounts ofthe ligand are titrated into the ITC cell, the quantity of heat absorbedor released is in direct proportion to the amount of binding. As thesystem reaches saturation, the heat signal diminishes until only heatsof dilution are observed. A binding curve is then obtained from a plotof the heats from each injection against the ratio of ligand and bindingpartner in the cell. The binding curve is analyzed with the appropriatebinding model to determine K_(B), n and ΔH. Note that K_(B)=1/K_(d).

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. In certain embodiments,subjects are “patients,” i.e., living humans that are receiving medicalcare for a disease or condition. This includes persons with no definedillness who are being investigated for signs of pathology. Preferred aresubjects who have an existing diagnosis of a particular cancer which isbeing targeted by the compositions and methods of the present invention.Preferred cancers for treatment with the compositions described hereininclude, but are not limited to prostate cancer, renal carcinoma,melanoma, pancreatic cancer, cervical cancer, ovarian cancer, coloncancer, head & neck cancer, lung cancer and breast cancer.

“Therapeutically effective amount” is defined as an amount of a reagentor pharmaceutical composition that is sufficient to show a patientbenefit, i.e., to cause a decrease, prevention, or amelioration of thesymptoms of the condition being treated. When the agent orpharmaceutical composition comprises a diagnostic agent, a“diagnostically effective amount” is defined as an amount that issufficient to produce a signal, image, or other diagnostic parameter.Effective amounts of the pharmaceutical formulation will vary accordingto factors such as the degree of susceptibility of the individual, theage, gender, and weight of the individual, and idiosyncratic responsesof the individual. “Effective amount” encompasses, without limitation,an amount that can ameliorate, reverse, mitigate, prevent, or diagnose asymptom or sign of a medical condition or disorder or a causativeprocess thereof. Unless dictated otherwise, explicitly or by context, an“effective amount” is not limited to a minimal amount sufficient toameliorate a condition.

“Treatment” or “treating” (with respect to a condition or a disease) isan approach for obtaining beneficial or desired results including andpreferably clinical results. For purposes of this invention, beneficialor desired results with respect to a disease include, but are notlimited to, one or more of the following: preventing a disease,improving a condition associated with a disease, curing a disease,lessening severity of a disease, delaying progression of a disease,alleviating one or more symptoms associated with a disease, increasingthe quality of life of one suffering from a disease, and/or prolongingsurvival. Likewise, for purposes of this invention, beneficial ordesired results with respect to a condition include, but are not limitedto, one or more of the following: preventing a condition, improving acondition, curing a condition, lessening severity of a condition,delaying progression of a condition, alleviating one or more symptomsassociated with a condition, increasing the quality of life of onesuffering from a condition, and/or prolonging survival. For instance, inembodiments where the compositions described herein are used fortreatment of cancer, the beneficial or desired results include, but arenot limited to, one or more of the following: reducing the proliferationof (or destroying) neoplastic or cancerous cells, reducing metastasis ofneoplastic cells found in cancers, shrinking the size of a tumor,decreasing symptoms resulting from the cancer, increasing the quality oflife of those suffering from the cancer, decreasing the dose of othermedications required to treat the disease, delaying the progression ofthe cancer, and/or prolonging survival of patients having cancer.Depending on the context, “treatment” of a subject can imply that thesubject is in need of treatment, e.g., in the situation where thesubject comprises a disorder expected to be ameliorated byadministration of a reagent.

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Immunomodulatory Cell Lines

By “inactivated tumor cell” is meant a tumor cell (either “autologous”or “allogeneic” to the patient) which has which been treated to preventdivision of the cells. For purposes of the present invention, such cellspreserve their immunogenicity and their metabolic activity. Such tumorcells are genetically modified to express a transgene which is expressedwithin a patient as part of cancer therapy. Thus, a composition orvaccine of the invention comprises neoplastic (e.g., tumor) cells thatare autologous or allogeneic to the patient undergoing treatment and ismost preferably the same general type of tumor cell as is afflicting thepatient. For example, a patient suffering from melanoma will typicallybe administered a genetically modified cell derived from a melanoma.Methods for inactivating tumor cells for use in the present invention,such as the use of irradiation, are well known in the art.

The inactivated tumor cells of the present invention are administered tothe patient together with one or more costimulatory molecules or agents.A preferred costimulatory agent comprises one or more cytokines whichstimulate dendritic cell induction, recruitment, and/or maturation.Methods for assessing such costimulatory agents are well known in theliterature. Induction and maturation of DCs is typically assessed byincreased expression of certain membrane molecules such as CD80 andCD86, and/or secretion of pro-inflammatory cytokines, such as IL-12 andtype I interferons following stimulation.

In preferred embodiments, the inactivated tumor cells themselves aremodified to express and secrete one or more cytokines which stimulatedendritic cell induction, recruitment, and/or maturation. The presentinvention is described in exemplary terms with regard to the use ofGM-CSF. Thus, by way of example, the tumor cell may express a transgeneencoding GM-CSF as described in U.S. Pat. Nos. 5,637,483, 5,904,920,6,277,368 and 6,350,445, as well as in US Patent Publication No.20100150946, each of which is expressly incorporated by referenceherein. A form of GM-CSF-expressing genetically modified cancer cells ora “cytokine-expressing cellular vaccine” for the treatment of pancreaticcancer is described in U.S. Pat. Nos. 6,033,674 and 5,985,290, both ofwhich are expressly incorporated by reference herein.

Other suitable cytokines which may be expressed by such inactivatedtumor cells and/or bystander cells instead of, or together with, GM-CSFinclude, but are not limited to, one or more of CD40 ligand, IL-12,CCL3, CCL20, and CCL21. This list is not meant to be limiting.

While it is preferred that the inactivated tumor cells administered tothe subject express one or more cytokines of interest, the tumor cellline may be accompanied by an inactivated bystander cell line whichexpresses and secretes one or more cytokines which stimulate dendriticcell induction, recruitment, and/or maturation. The bystander cell linemay provide all of the cytokines which stimulate dendritic cellinduction, recruitment, and/or maturation, or may supplement cytokineswhich stimulate dendritic cell induction, recruitment, and/or maturationexpressed and secreted by the inactivated tumor cells. By way ofexample, immunomodulatory cytokine-expressing bystander cell lines aredisclosed in U.S. Pat. Nos. 6,464,973, and 8,012,469, Dessureault etal., Ann. Surg. Oncol. 14: 869-84, 2007, and Eager and Nemunaitis, Mol.Ther. 12: 18-27, 2005, each of which is expressly incorporated byreference herein.

By “Granulocyte-macrophage colony stimulating factor (GM-CSF)polypeptide” is meant a cytokine or fragment thereof havingimmunomodulatory activity and having at least about 85% amino acidsequence identity to GenBank Accession No. AAA52122.1.

Cyclic Purine Dinucleotides

As described herein, another of these costimulatory agents is a cyclicpurine dinucleotide which binds to STING and induces STING-dependentTBK1 activation. Other costimulatory molecules which may be included aredescribed hereinafter.

Prokaryotic as well as eukaryotic cells use various small molecules forcell signaling and intra- and intercellular communication. Cyclicnucleotides like cGMP, cAMP, etc. are known to have regulatory andinitiating activity in pro- and eukaryotic cells. Unlike eukaryoticcells, prokaryotic cells also use cyclic purine dinucleotides asregulatory molecules. In prokaryotes, the condensation of two GTPmolecules is catalyst by the enzyme diguanylate cyclase (DGC) to givec-diGMP, which represents an important regulator in bacteria.

Recent work suggests that cyclic diGMP or analogs thereof can alsostimulate or enhance immune or inflammatory response in a patient or canenhance the immune response to a vaccine by serving as an adjuvant inmammals. Cytosolic detection of pathogen-derived DNA requires signalingthrough TANK binding kinase 1 (TBK1) and its downstream transcriptionfactor, IFN-regulatory factor 3 (IRF3). A transmembrane protein calledSTING (stimulator of IFN genes; also known as MITA, ERIS, MPYS andTMEM173) functions as the signaling receptor for these cyclic purinedinucleotides, causing stimulation of the TBK1-IRF3 signalling axis anda STING-dependent type I interferon response. See, e.g., FIG. 1.Burdette et al., Nature 478: 515-18, 2011 demonstrated that STING bindsdirectly to cyclic diguanylate monophosphate, but not to other unrelatednucleotides or nucleic acids.

Suitable cyclic purine dinucleotides for use in the present inventionare described in some detail in, e.g., U.S. Pat. Nos. 7,709,458 and7,592,326; WO2007/054279; and Yan et al., Bioorg. Med. Chem. Lett. 18:5631 (2008), each of which is hereby incorporated by reference.Preferred cyclic purine dinucleotides include, but are not limited to,c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP, c-AMP-IMP, and c-GMP-IMP, andanalogs thereof including, but not limited to, phosphorothioateanalogues.

Adjuvants

In addition to the inactivated tumor cell(s) and cyclic purinedinucleotide(s) described above, the compositions of the presentinvention may further comprise one or more additional substances which,because of their adjuvant nature, can act to stimulate the immune systemto respond to the cancer antigens present on the inactivated tumorcell(s). Such adjuvants include, but are not limited to, lipids,liposomes, inactivated bacteria which induce innate immunity (e.g.,inactivated or attenuated Listeria monocytogenes), compositions whichmediate innate immune activation via Toll-like Receptors (TLRs),(NOD)-like receptors (NLRs), Retinoic acid inducible gene-based(RIG)-1-like receptors (RLRs), and/or C-type lectin receptors (CLRs).Examples of PAMPs include lipoproteins, lipopolypeptides,peptidoglycans, zymosan, lipopolysaccharide, neisserial porins,flagellin, profillin, galactoceramide, muramyl dipeptide.Peptidoglycans, lipoproteins, and lipoteichoic acids are cell wallcomponents of Gram-positive. Lipopolysaccharides are expressed by mostbacteria, with MPL being one example. Flagellin refers to the structuralcomponent of bacterial flagella that is secreted by pathogenic andcommensal bacterial. α-Galactosylceramide (α-GalCer) is an activator ofnatural killer T (NKT) cells. Muramyl dipeptide is a bioactivepeptidoglycan motif common to all bacteria. This list is not meant to belimiting. Preferred adjuvant compositions are described below.

CTLA-4 and PD-1 Pathway Antagonists

CTLA-4 is thought to be an important negative regulator of the adaptiveimmune response. Activated T cells upregulate CTLA-4, which binds CD80and CD86 on antigen-presenting cells with higher affinity than CD28,thus inhibiting T-cell stimulation, IL-2 gene expression and T-cellproliferation. Anti-tumor effects of CTLA4 blockade have been observedin murine models of colon carcinoma, metastatic prostate cancer, andmetastatic melanoma.

Ipilimumab (Yervoy™) and tremelimumab are humanized monoclonalantibodies that bind to human CTLA4 and prevent its interaction withCD80 and CD86. Phase I and II studies using ipilimumab and tremelimumabhave demonstrated clinical activity in cancer patients. Other negativeimmune regulators which may be targeted by a similar strategy includeprogrammed cell death 1, B and T lymphocyte attenuator, transforminggrowth factor beta β, interleukin-10, and vascular endothelial growthfactor.

PD-1 is another negative regulator of adaptive immune response that isexpressed on activated T-cells. PD-1 binds to B7-H1 and B7-DC, and theengagement of PD-1 suppresses T-cell activation. Anti-tumor effects havebeen demonstrated with PD-1 pathway blockade. BMS-936558, MK3475,CT-011, AMP-224 and MDX-1106 have been reported in the literature to beexamples of PD-1 pathway blockers which may find use in the presentinvention.

TLR Agonists

The term “Toll like receptor” (or “TLR”) as used herein refers to amember of the Toll-like receptor family of proteins or a fragmentthereof that senses a microbial product and/or initiates an adaptiveimmune response. In one embodiment, a TLR activates a dendritic cell(DC). Toll like receptors (TLRs) are a family of pattern recognitionreceptors that were initially identified as sensors of the innate immunesystem that recognize microbial pathogens. TLRs comprise a family ofconserved membrane spanning molecules containing an ectodomain ofleucine-rich repeats, a transmembrane domain and an intracellular TIR(Toll/IL-1R) domain. TLRs recognize distinct structures in microbes,often referred to as “PAMPs” (pathogen associated molecular patterns).Ligand binding to TLRs invokes a cascade of intra-cellular signalingpathways that induce the production of factors involved in inflammationand immunity.

In humans, ten TLR have been identified. TLRs that are expressed on thesurface of cells include TLR-1, -2, -4, -5, and -6, while TLR-3, -7/8,and -9 are expressed with the ER compartment. Human dendritic cellsubsets can be identified on the basis of distinct TLR expressionpatterns. By way of example, the myeloid or “conventional” subset of DC(mDC) expresses TLRs 1-8 when stimulated, and a cascade of activationmarkers (e.g. CD80, CD86, MHC class I and II, CCR7), pro-inflammatorycytokines, and chemokines are produced. A result of this stimulation andresulting expression is antigen-specific CD4+ and CD8+ T cell priming.These DCs acquire an enhanced capacity to take up antigens and presentthem in an appropriate form to T cells. In contrast, the plasmacytoidsubset of DC (pDC) expresses only TLR7 and TLR9 upon activation, with aresulting activation of NK cells as well as T-cells. As dying tumorcells may adversely affect DC function, it has been suggested thatactivating DC with TLR agonists may be beneficial for priming anti-tumorimmunity in an immunotherapy approach to the treatment of cancer. It hasalso been suggested that successful treatment of breast cancer usingradiation and chemotherapy requires TLR4 activation.

TLR agonists known in the art and finding use in the present inventioninclude, but are not limited to, the following:

Pam3Cys, a TLR-1/2 agonist;

CFA, a TLR-2 agonist;

MALP2, a TLR-2 agonist;

Pam2Cys, a TLR-2 agonist;

FSL-1, a TLR-2 agonist;

Hib-OMPC, a TLR-2 agonist;

polyribosinic:polyribocytidic acid (Poly I:C), a TLR-3 agonist;

polyadenosine-polyuridylic acid (poly AU), a TLR-3 agonist;

Polyinosinic-Polycytidylic acid stabilized with poly-L-lysine andcarboxymethylcellulose (Hiltonol®), a TLR-3 agonist;

monophosphoryl lipid A (MPL), a TLR-4 agonist;

LPS, a TLR-4 agonist;

bacterial flagellin, a TLR-5 agonist;

sialyl-Tn (STn), a carbohydrate associated with the MUC1 mucin on anumber of human cancer cells and a TLR-4 agonist;

imiquimod, a TLR-7 agonist;

resiquimod, a TLR-7/8 agonist;

loxoribine, a TLR-7/8 agonist; and

unmethylated CpG dinucleotide (CpG-ODN), a TLR-9 agonist.

Because of their adjuvant qualities, TLR agonists are preferably used incombinations with other vaccines, adjuvants and/or immune modulators,and may be combined in various combinations. Thus, in certainembodiments, the cyclic purine dinucleotides that bind to STING andinduces STING-dependent TBK1 activation and an inactivated tumor cellwhich expresses and secretes one or more cytokines which stimulatedendritic cell induction, recruitment and/or maturation, as describedherein can be administered together with one or more TLR agonists fortherapeutic purposes.

Lipids and Liposomes

Liposomes are vesicles formed from one (“unilamellar”) or more(“multilamellar”) layers of phospholipid. Because of the amphipathiccharacter of the phospholipid building blocks, liposomes typicallycomprise a hydrophilic layer presenting a hydrophilic external face andenclosing a hydrophilic core. The versatility of liposomes in theincorporation of hydrophilic/hydrophobic components, their non-toxicnature, biodegradability, biocompatibility, adjuvanticity, induction ofcellular immunity, property of sustained release and prompt uptake bymacrophages, makes them attractive candidates for the delivery ofantigens.

WO2010/104833, which is incorporated by reference herein in itsentirety, describes liposomal preparations which comprise:

-   -   a) an aqueous vehicle;    -   b) liposomes comprising    -   (i) dimyristoylphosphatidylcholine (“DMPC”),    -   (ii) dimyristoylphosphatidylglycerol (“DMPG”),        dimyristoyltrimethylammonium propane (“DMTAP”), or both DMPG and        DMTAP, and    -   (iii) at least one sterol derivative; and    -   c) one or more immunogenic polypeptide(s) or carbohydrate(s)        covalently linked to between 1% and 100% of said at least one        sterol derivative.

Such liposomal formulations, referred to herein as VesiVax® (MolecularExpress, Inc.), with our without the “immunogenic polypeptide(s) orcarbohydrate(s)” referred to above, can contain one or more additionalcomponents such as peptidoglycan, lipopeptide, lipopolysaccharide,monophosphoryl lipid A, lipoteichoic acid, resiquimod, imiquimod,flagellin, oligonucleotides containing unmethylated CpG motifs,beta-galactosylceramide, muramyl dipeptide, all-trans retinoic acid,double-stranded viral RNA, heat shock proteins,dioctadecyldimethylammonium bromide, cationic surfactants, toll-likereceptor agonists, dimyristoyltrimethylammoniumpropane, and nod-likereceptor agonists. Advantageously, these liposomal formulations can beused to deliver one or more cyclic purine dinucleotides in accordancewith the present invention.

Moreover, while the liposomal formulations discussed above employ a“steroid derivative” as an anchor for attaching an immunogenicpolypeptide or carbohydrate to a liposome, the steroid may simply beprovided as an unconjugated steroid such as cholesterol.

Suitable methods for preparing liposomes from lipid mixtures are wellknown in the art. See, e.g., Basu & Basu, Liposome Methods and Protocols(Methods in Molecular Biology), Humana Press, 2002; Gregoriadis,Liposome Technology, 3^(rd) Edition, Informa HealthCare, 2006. Preferredmethods include extrusion, homogenization, and sonication methodsdescribed therein. An exemplary method for preparing liposomes for usein the present invention, which comprises drying a lipid mixture,followed by hydration in an aqueous vehicle and sonication to formliposomes, is described in WO2010/104833.

In certain embodiments, the liposomes are provided within a particularaverage size range. Liposome size can be selected, for example, byextrusion of an aqueous vehicle comprising liposomes through membraneshaving a preselected pore size and collecting the material flowingthrough the membrane. In preferred embodiments, the liposomes areselected to be substantially between 50 and 500 nm in diameter, morepreferably substantially between 50 and 200 nm in diameter, and mostpreferably substantially between 50 and 150 nm in diameter. The term“substantially” as used herein in this context means that at least 75%,more preferably 80%, and most preferably at least 90% of the liposomesare within the designated range.

Other lipid and lipid-like adjuvants which may find use in the presentinvention include oil-in-water (o/w) emulsions (see, e.g., Muderhwa etal., J. Pharmaceut. Sci. 88: 1332-9, 1999)), VesiVax® TLR (MolecularExpress, Inc.), digitonin (see, e.g., U.S. Pat. No. 5,698,432), andglucopyranosyl lipids (see, e.g., United States Patent Application20100310602).

Chemotherapeutic Agents

In additional embodiments the methods further involve administering tothe subject an effective amount of one or more chemotherapeutics as anadditional treatment for the patient's tumor. In certain embodiments theone or more chemo therapeutics is selected from abiraterone acetate,altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide,BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, bleomycin,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide,cachectin, cemadotin, chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol,doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide,hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine,lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard),melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin,mitomycin, methotrexate, taxanes, nilutamide, onapristone, paclitaxel,prednimustine, procarbazine, RPR109881, stramustine phosphate,tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine,vindesine sulfate, and vinflunine.

Pharmaceutical Compositions

The term “pharmaceutical” as used herein refers to a chemical substanceintended for use in the cure, treatment, or prevention of disease andwhich is subject to an approval process by the U.S. Food and DrugAdministration (or a non-U.S. equivalent thereof) as a prescription orover-the-counter drug product. Details on techniques for formulation andadministration of such compositions may be found in Remington, TheScience and Practice of Pharmacy 21^(st) Edition (Mack Publishing Co.,Easton, Pa.) and Nielloud and Marti-Mestres, Pharmaceutical Emulsionsand Suspensions: 2^(nd) Edition (Marcel Dekker, Inc, New York).

For the purposes of this disclosure, the pharmaceutical compositions maybe administered by a variety of means including orally, parenterally, byinhalation spray, topically, or rectally in formulations containingpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used here includes but is not limited to subcutaneous,intravenous, intramuscular, intraarterial, intradermal, intrathecal andepidural injections with a variety of infusion techniques. Intraarterialand intravenous injection as used herein includes administration throughcatheters. Administration via intracoronary stents and intracoronaryreservoirs is also contemplated. The term oral as used herein includes,but is not limited to oral ingestion, or delivery by a sublingual orbuccal route. Oral administration includes fluid drinks, energy bars, aswell as pill formulations.

Pharmaceutical compositions may be in any form suitable for the intendedmethod of administration. When used for oral use for example, tablets,troches, lozenges, aqueous or oil suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups or elixirs may beprepared. Compositions intended for oral use may be prepared accordingto any method known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation. Tabletscontaining a drug compound in admixture with non-toxic pharmaceuticallyacceptable excipient which are suitable for manufacture of tablets areacceptable. These excipients may be, for example, inert diluents, suchas calcium or sodium carbonate, lactose, calcium or sodium phosphate;granulating and disintegrating agents, such as maize starch, or alginicacid; binding agents, such as starch, gelatin or acacia; and lubricatingagents; such as magnesium stearate, stearic acid or talc. Tablets may beuncoated, or may be coated by known techniques including entericcoating, colonic coating, or microencapsulation to delay disintegrationand adsorption in the gastrointestinal tract and/or provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the drug compound is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Pharmaceutical compositions may be formulated as aqueous suspensions inadmixture with excipients suitable for the manufacture ofaqueous-suspensions. Such excipients include a suspending agent, such assodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanthand gum acacia, and dispersing or wetting agents such as a naturallyoccurring phosphatide (e.g., lecithin), a condensation product of analkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), acondensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or a mineral oil such as liquid paraffin. The oral suspensions maycontain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the disclosure suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the disclosure may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oil,such as olive oil or arachis oil, a mineral oil, such as liquidparaffin, or a mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the disclosure may be in the form ofa sterile injectable preparation, such as a sterile injectable aqueousor oleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solventsuch as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 20 to 500 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions. It is preferredthat the pharmaceutical composition be prepared which provides easilymeasurable amounts for administration. Typically, an effective amount tobe administered systemically is about 0.1 mg/kg to about 100 mg/kg anddepends upon a number of factors including, for example, the age andweight of the subject (e.g., a mammal such as a human), the precisecondition requiring treatment and its severity, the route ofadministration, and will ultimately be at the discretion of theattendant physician or veterinarian. It will be understood, however,that the specific dose level for any particular patient will depend on avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex and diet of theindividual being treated; the time and route of administration; the rateof excretion; other drugs which have previously been administered; andthe severity of the particular condition undergoing therapy, as is wellunderstood by those skilled in the art.

As noted above, formulations of the disclosure suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient, as a powder or granules; as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The pharmaceutical compositions may alsobe administered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in a freeflowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropyl ethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) surface active or dispersing agent. Moldedtablets may be made in a suitable machine using a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropyl methylcellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric or colonic coating to provide release in partsof the gut other than the stomach. This is particularly advantageouswith the compounds of formula 1 when such compounds are susceptible toacid hydrolysis.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin, or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

As used herein, pharmaceutically acceptable salts include, but are notlimited to: acetate, pyridine, ammonium, piperazine, diethylamine,nicotinamide, formic, urea, sodium, potassium, calcium, magnesium, zinc,lithium, cinnamic, methylamino, methanesulfonic, picric, tartaric,triethylamino, dimethylamino, and tris(hydroxymethyl)aminomethane.Additional pharmaceutically acceptable salts are known to those skilledin the art.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the route and dose of administration and the severity of sideeffects. Guidance for methods of treatment and diagnosis is available(see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good ClinicalPractice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

An effective amount may be given in one dose, but is not restricted toone dose. Thus, the administration can be two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, twenty, or more, administrationsof pharmaceutical composition. Where there is more than oneadministration of a pharmaceutical composition in the present methods,the administrations can be spaced by time intervals of one minute, twominutes, three, four, five, six, seven, eight, nine, ten, or moreminutes, by intervals of about one hour, two hours, three, four, five,six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 hours, and so on. In the context of hours, the term“about” means plus or minus any time interval within 30 minutes. Theadministrations can also be spaced by time intervals of one day, twodays, three days, four days, five days, six days, seven days, eightdays, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days,16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinationsthereof. The invention is not limited to dosing intervals that arespaced equally in time, but encompass doses at non-equal intervals.

A dosing schedule of, for example, once/week, twice/week, threetimes/week, four times/week, five times/week, six times/week, seventimes/week, once every two weeks, once every three weeks, once everyfour weeks, once every five weeks, and the like, is available for theinvention. The dosing schedules encompass dosing for a total period oftime of, for example, one week, two weeks, three weeks, four weeks, fiveweeks, six weeks, two months, three months, four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, and twelve months.

Provided are cycles of the above dosing schedules. The cycle can berepeated about, e.g., every seven days; every 14 days; every 21 days;every 28 days; every 35 days; 42 days; every 49 days; every 56 days;every 63 days; every 70 days; and the like. An interval of non dosingcan occur between a cycle, where the interval can be about, e.g., sevendays; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63days; 70 days; and the like. In this context, the term “about” meansplus or minus one day, plus or minus two days, plus or minus three days,plus or minus four days, plus or minus five days, plus or minus sixdays, or plus or minus seven days.

Methods for co-administration with an additional therapeutic agent arewell known in the art (Hardman, et al. (eds.) (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, 10th ed.,McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001)Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.).

As noted, the compositions of the present invention are preferablyformulated as pharmaceutical compositions for parenteral or enteraldelivery. A typical pharmaceutical composition for administration to ananimal comprises a pharmaceutically acceptable vehicle such as aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like. See, e.g., Remington's Pharmaceutical Sciences, 15th Ed.,Easton ed., Mack Publishing Co., pp 1405-1412 and 1461-1487 (1975); TheNational Formulary XIV, 14th Ed., American Pharmaceutical Association,Washington, D.C. (1975). Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil and injectable organic esterssuch as ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to routine skills in the art.

Repeated administrations of a particular vaccine (homologous boosting)have proven effective for boosting humoral responses. Such an approachmay not be effective at boosting cellular immunity because priorimmunity to the vector tends to impair robust antigen presentation andthe generation of appropriate inflammatory signals. One approach tocircumvent this problem has been the sequential administration ofvaccines that use different antigen-delivery systems (heterologousboosting). In a heterologous boosting regimen, at least one prime orboost delivery comprises delivery of the inactivated tumor cell/cyclicpurine dinucleotide compositions described herein. The heterologous armof the regimen may comprise delivery of antigen using one or more of thefollowing strategies:

-   -   inactivated bacteria or viruses comprising the antigen of        interest, which are particles that have been treated with some        denaturing condition to render them ineffective or inefficient        in mounting a pathogenic invasion;    -   purified antigens, which are typically naturally-produced        antigens purified from a cell culture of the pathogen or a        tissue sample containing the pathogen, or a recombinant version        thereof;    -   live viral or bacterial delivery vectors recombinantly        engineered to express and/or secrete antigens in the host cells        of the subject. These strategies rely on attenuating (e.g., via        genetic engineering) the viral or bacterial vectors to be        non-pathogenic and non-toxic;    -   antigen presenting cell (APC) vectors, such as a dendritic        cell (DC) vector, which comprise cells that are loaded with an        antigen, or transfected with a composition comprising a nucleic        acid encoding the antigen (e.g., Provenge® (Dendreon        Corporation) for the treatment of castration-resistant        metastatic prostate cancer);    -   liposomal antigen delivery vehicles; and    -   naked DNA vectors and naked RNA vectors which may be        administered by a gene gun, electroporation, bacterial ghosts,        microspheres, microparticles, liposomes, polycationic        nanoparticles, and the like.

A prime vaccine and a boost vaccine can be administered by any one orcombination of the following routes. In one aspect, the prime vaccineand boost vaccine are administered by the same route. In another aspect,the prime vaccine and boost vaccine are administered by differentroutes. The term “different routes” encompasses, but is not limited to,different sites on the body, for example, a site that is oral, non-oral,enteral, parenteral, rectal, intranode (lymph node), intravenous,arterial, subcutaneous, intramuscular, intratumor, peritumor,intratumor, infusion, mucosal, nasal, in the cerebrospinal space orcerebrospinal fluid, and so on, as well as by different modes, forexample, oral, intravenous, and intramuscular.

An effective amount of a prime or boost vaccine may be given in onedose, but is not restricted to one dose. Thus, the administration can betwo, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more, administrations of the vaccine. Where there is morethan one administration of a vaccine the administrations can be spacedby time intervals of one minute, two minutes, three, four, five, six,seven, eight, nine, ten, or more minutes, by intervals of about onehour, two hours, three, four, five, six, seven, eight, nine, ten, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. Inthe context of hours, the term “about” means plus or minus any timeinterval within 30 minutes. The administrations can also be spaced bytime intervals of one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, and combinations thereof. The invention is not limited todosing intervals that are spaced equally in time, but encompass doses atnon-equal intervals, such as a priming schedule consisting ofadministration at 1 day, 4 days, 7 days, and 25 days, just to provide anon-limiting example.

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EXAMPLES

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1

One approach to stimulate immunity against a broad repertoire of TAAs is“GVAX,” which are vaccines based on allogeneic human tumor cell linesthat are engineered to secrete GM-CSF, the primary cytokine thatstimulates DC recruitment, differentiation and maturation. GVAX vaccineshave formed the basis of multiple clinical trials in several cancerindications, and have been shown to be safe, well-tolerated, immunogenicand shown to provide some clinical benefit. A Phase 3 clinical studycomparing prostate GVAX immunotherapy (G) to docetaxel plus prednisone(D+P) in men with mCRPC was prematurely terminated by the sponsor whenan early unscheduled futility analysis revealed that the trial had <30%chance of meeting its predefined primary endpoint of improvement inoverall survival. Continued follow-up and analysis of the >600 patientson study, however, revealed that the Kaplan-Meier survival curve for theG arm crossed above the D+P arm at ˜21 months. Furthermore, patientswith a predicted survival time of ≧18 months at baseline, based on theHalabi nomogram, had a 2.5 month survival benefit when treated withGVAX, with a 30% “tail” of long-term survivors, as compared tochemotherapy These results showed that GVAX prostate immunotherapyprovided a survival benefit over chemotherapy, which correspondinglyshows ˜2 month survival benefit as compared to placebo.

Since TLR-targeted adjuvants used individually that signal throughMyD88- and TRIF-dependent pathways are typically poor inducers of CD8 Tcell immunity, we assessed whether CDN, which signals through thecytoplasmic STING receptor, could facilitate priming of both MHC classI- and class II-restricted immunity. CDN induced priming of both Th1 CD4and CD8 T cells specific for the vaccine recombinant protein Ag, HIV Gagor OVA. Balb/c mice were vaccinated twice subcutaneously in the base ofthe tail (s.c.) 3 wks apart with 5 μg of HIV Gag protein formulated with2% oil-in-water adjuvant (Addavax, Invivogen) and CDN at the dose levelindicated in FIG. 2.

As shown in FIG. 2A, CDN-adjuvanted HIV Gag vaccines induce apolyfunctional Ag-specific Th1 CD4 T cell response. The secondary CD4 Tcell response was measured at 5 days post boost by intracellularcytokine staining of IFN-γ, IL-2 and TNF-α positive splenocytesfollowing stimulation with the I-A^(d) restricted HIV Gag epitope. Barsrepresent individual mice. As shown in FIG. 4B, the magnitude of theCDN-dependent vaccine induced T cell response is enhanced by formulationof CDN with VesiVax® liposomes.

The primary (1) and secondary (2) OVA-specific CD4 and CD8 T cellresponse in PBMC following s.c. immunization of groups of 5 C57BL/6 micewith the vaccine compositions shown in FIG. 2 was measured by IFN-γELISPOT, and the results depicted in FIG. 2C. Groups of 5 C57BL/6 micewere immunized s.c. twice at a 3-wk interval with the vaccines indicatedin the Figure, or with 5×10⁶CFU of Lm-OVA, given i.v. At 4 weeks postboost, mice were challenged with 5×10⁵ PFU of VV-OVA, and 5 days laterthe ovaries were harvested, processed and VV-OVA was quantitated byplaque assay.

As shown in the figure, vaccine potency was dependent on formulation,and our initial results indicate that formulation with VesiVax®liposomes was optimal, due likely to efficient vaccine delivery into thecytosol. Notably, mice vaccinated with CDN-adjuvanted OVA werecompletely protected by challenge with vaccinia virus. By comparisonwith the negative control group given HBSS, this level of protectionwas >4 logs. The level of protection afforded by the CDN-adjuvantedvaccine was better than either MPL-adjuvanted OVA (using the human MPLdose of 50 μg), or Listeria-OVA vaccines.

FIG. 7 depicts the induction of human dendritic cells upon CDNtreatment, as assessed by expression of costimulatory molecules such asCD80 and CD86. In this experiment, CD14+ monocytes were isolated fromPBMC and cultured in the presence of GM-CSF and IL-4. On day 6, 10⁵ DCswere treated with 100 ng/ML LPS, 20 μM CDN (c-di-AMP); 369 μg liposomes(VesiVax®), or liposomes plus CDN, as indicated in the figure. Theindicated costimulatory molecules were detected 48 hrs later by flowcytometry. As noted, liposomes substantially improve the ability of CDNto induce dendritic cell maturation.

Example 2

The B16 melanoma tumor model is aggressive and poorly immunogenic, andtherapeutic vaccination with irradiated GM-CSF secreting B16 melanomatumor cells (B16-GM) has not been effective unless combined withblockade of immune checkpoints, such as CTLA-4 or PD-1. In this example,we show that a single injection of STINGVAX (CDN formulated withdigitonin and incubated with irradiated B16-GM) significantly inhibitedthe growth of established palpable B16 tumors, when administered at 7days post B16 tumor cell implantation when the tumor was palpable andestablished.

5×10⁴ B16 melanoma cells were inoculated in the footpad of C57BL/6 miceand when tumors were palpable at 7 days, mice were injected once s.c. inthe contralateral thigh with the vaccines indicated in FIG. 3. Thefollowing amounts of vaccine components were used for each injection:irradiated B16 GM-CSF (GVAX), 1.5×10⁶ cells; CDN only, 20 ng; digitonin,10 μg/mL. STINGVAX was prepared by incubation of irradiated B16 GM-CSFwith CDN and digitonin at 20° C. for 30 min, washing 3× with PBS, andthe resulting composition injected after resuspension in 200 μL of PBS.See also Woodward et al., Supporting online material 27 May 2010 onScience Express DOI: 10.1126/science. 1189801, for formulationinformation. Tumor growth was measured daily. Growth was significantlyinhibited by STINGVAX vs No Rx Group (P<0.01). As shown in FIG. 3A,tumor inhibition was dependent on combination of B16-GM with CDN, astreatment with either component alone had no impact on tumor growth ascompared to untreated control mice. Additionally, the CD8 T cellresponse specific for p15E, an endogenous retrovirus-specific Agexpressed on the B16 tumor, was enhanced in STINGVAX-treatedtumor-bearing mice, compared to B16-GM treated mice (data not shown).

From these data, it is believed that the synergy between STING-targetedCDN and irradiated GM-secreting tumor cell vaccines requires that theCDN-dependent induction of IFN-β results in the activation ofGM-CSF-differentiated DCs, thus providing a strong “Signal 3,” resultingin a more-effective antitumor T cell response. For this reason, wedetermined whether CDN could directly induce the production of IFN-β inTRAMP-GM cells. As shown in FIG. 3B, when formulated with digitonin, CDNefficiently induced the expression of IFN-β in both TRAMP-GM and primarymacrophages, but not in macrophages from goldenticket (gt) mice whichlack a functional STING protein.

FIG. 4 demonstrates the dose-dependency of the therapeutic benefitexhibited by the STING-targeted CDN/GVAX combination. In thisexperiment, 5×10⁴ B16 melanoma cells were inoculated in the footpad ofC57BL/6 mice. Once tumor is palpable at day 6, GVAX (B16 GM-CSF (GVAX),1.5×10⁶ cells) or the CDN/GVAX combination at 2, 20, or 200 ngCDN/animal was injected subcutaneously into the contralateral thigh. Inthe case of the combination treatment, the CDN was formulated withdigitonin at 10 μg/mL for 30 min at 20° C. and subsequently washed 4×with PBS to remove non-incorporated CDN prior to injection as describedabove. Tumor volume was measured daily in a total of 20animals/treatment group. As shown in the figure, the antitumor responsewas increased as the concentration of CDN increased from 2 ng/animal to20 ng/animal, but no further increase was observed by increasing thedosage to 200 ng/mL.

FIG. 5 provides further evidence of a synergistic antitumor response tothe CDN/GVAX combination. In this figure, B16 melanoma cells wereharvested from the animals and the cells fixed onto slides forimmunohistochemistry. Fluorescein-labeled anti-CD8 antibodies were isedto visualize CR8+ T-cell infiltration into the tumor. DAPI was used tocounterstain the nucleus of the tumor cells. The four panels shown inthe figure are untreated B16 melanoma cells (A), and cells treated withCDN (B), GVAX (C) and CDN/GVAX (D). As shown, a substantial improvementin CD*+ T-cell tumor infiltration is observed with CDN/GVAX incomparison to either of CDN or GVAX alone.

Similarly, FIG. 6 demonstrates an improved induction of matureinterferon γ-producing splenic DC (CD11c+ cells) by CDN/GVAX incomparison to either of CDN or GVAX alone. In this experiment, mice weretreated as described above, and the spleen from 2 mice/group wereharvested. Total splenocytes were harvested and stained with anti-CD11cand anti-IFNa conjugates. The treatment (untreated B16, CDN,CDN+digitonin, GVAX, and CDN/GVAX+digitonin) are indicated in thefigure.

Example 3

FIG. 9 depicts the synergistic mechanism of action of “STINGVAX,” whichis STING-activating cyclic purine dinucleotides co-formulated withirradiated GM-CSF expressing allogeneic tumor cells (GVAX;STING+GVAX=STINGVAX). The GVAX tumor cell vaccines provides an un-biasedpresentation of multiple tumor associated antigens to the immune system.GM-CSF produced by GVAX recruits dendritic cells (DCs) to the injectionsite. CDNs activate the recruited DCs, which in turn activate or primepotent antigen-specific CD4 and CD8 T cells that traffic to and kill thetumor, resulting in a clinical benefit. STINGVAX can enhance the tumorresponse either by serving as a depot for the GM-CSF recruited DCs, orthrough autocrine signaling express both TBK-1/IRF-3 dependent IFN-β andNF-κB pro-inflammatory cytokines that in combination activate the GM-CSFrecruited DCs.

The mechanism for the increased anti-tumor efficacy of STINGVAX isbelieved due to the activation of innate immunity that is mediated bydirect binding of CDNs to STING, triggering a conformational change inthis receptor, resulting in signaling through the TBK-1/IRF-3 axis andactivation of type 1 interferons (IFN), including IFN-α and IFN-β.GM-CSF produced by the GVAX tumor cell vaccine recruits dendritic cells(DCs) to the injection site. CDNs activate the recruited DCs, which inturn activate or prime potent antigen-specific CD4 and CD8 T cells thattraffic to and kill the tumor, resulting in a clinical benefit.

To determine the levels of IFN-α as a signature of CDN potency toactivate innate immunity, 1×10⁶ primary human PBMCs isolated fromfifteen independent human donors were incubated in a 96 well U bottomplate for 30 min at 37° C., 5% CO2 with 50 μM of c-di-GMP (CDG), 1 μg/mLof Interferon Stimulatory DNA (ISD), or 4 μg/mL of Poly (I:C) utilizingEffectene transfection reagent (Qiagen) to transfer the molecules intothe PBMC. ISD (Interferon Stimulating DNA) is TLR independent (Stetston,D. B. et. al. Immunity 24, 93-103, January 2006) and signals throughcGAS, and is thus STING-dependent, while Poly (I:C) can signal throughboth TLR3 and RIG-I pathways, and are thus STING-independent. After 30minutes, the cells were washed and replaced with RPMI media containing10% FBS and incubated at 37° C., 5% CO2. After 24 hours incubation IFN-αlevels were determined by Cytometric Bead Array (CBA, BD Biosciences)(FIG. 8). These results demonstrate that cyclic-di-GMP activates innateimmunity in human leukocytes prepared from multiple independent donors,thus supporting the mechanism of action of STINGVAX.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

It is to be understood that the invention is not limited in itsapplication to the details of construction and to the arrangements ofthe components set forth in the following description or illustrated inthe drawings. The invention is capable of embodiments in addition tothose described and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein, as well as the abstract, are for the purpose ofdescription and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

The invention claimed is:
 1. A composition comprising: a cyclic purinedinucleotide which binds to STING and induces STING-dependent TBK1activation; and an inactivated tumor cell which expresses and secretesgranulocyte-macrophage colony-stimulating factor (GM-CSF).
 2. Acomposition according to claim 1, further comprising a pharmaceuticallyacceptable excipient.
 3. A composition according to claim 1, wherein thetumor cell is inactivated by treatment with radiation.
 4. A compositionaccording to claim 1, wherein the cyclic purine dinuclotide is selectedfrom the group consisting of c-di-AMP, c-di-GMP, c-di-IMP, c-AMP-GMP,c-AMP-IMP, and c-GMP-IMP, or combinations thereof.
 5. A compositionaccording to claim 1, wherein the cyclic purine dinuclotide isformulated with one or more lipids.
 6. A composition according to claim5, wherein the cyclic purine dinuclotide is formulated with digitonin.7. A composition according to claim 5, wherein the one or more lipidsform a liposome.
 8. A composition according to claim 1, furthercomprising one or more adjuvants.
 9. A composition according to claim 8,wherein the one or more adjuvants comprise CpG and/or monophosphoryllipid A.